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Applied and Environmental Microbiology, August 2004, p. 4996-5004, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4996-5004.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Joanna Peltola,^1* Alberto Ritieni,² Raimo Mikkola,¹ Pavel A. Grigoriev,^1,3 Gabriella Pócsfalvi,⁴ Maria A. Andersson,¹ and Mirja S. Salkinoja-Salonen¹

Division of Microbiology, Department of Applied Chemistry and Microbiology, University of Helsinki, FIN-00014 Helsinki, Finland,¹ Department of Food Science, "Federico II" Naples University, 80055 Portici,² Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science and Technology, CNR, 83100 Avellino, Italy,⁴ Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia³

Received 19 December 2003/ Accepted 27 April 2004

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 µg [dry weight] ml of extended boar semen^–1). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.

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* Corresponding author. Present address: Department of Applied Chemistry and Microbiology, Division of Microbiology, P.O. Box 56, FIN-00014 University of Helsinki, Finland. Phone: 358-(0)50-3392283. Fax: 358-(0)9-19159322. E-mail: JOANNA.PELTOLA{at}PP.INET.FI.

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Applied and Environmental Microbiology, August 2004, p. 4996-5004, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4996-5004.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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